1. Field of the Invention
The invention relates generally to the field of molecular biology. More specifically, it relates to compositions and methods for modulating and analyzing gene function in plants.
2. Description of the Related Art
Several technologies have been used to determine plant gene function in vivo. For example, classical breeding of cultivars allows the genetic mapping of various genes. Mutagenesis of plants followed by analysis of progeny identifies gene function through loss of specific phenotypes. Transformation of plants with sequences of unknown function followed by phenotype analysis of progeny is another example of a technology used by research scientists to determine gene function. However, these techniques require a large amount of time to obtain results.
Recently, a new procedure for identifying gene function in plants has appeared and captured the interests of many plant scientists. This procedure utilizes plant viruses to express a small portion of host genes with unknown functions in the infected plant. The replication of the virus vector induces a host surveillance system that will knock out expression of genes with identity to the transiently expressed sequence through the mechanism known as virus-induced gene silencing (VIGS) (Baulcombe, 1999; Vance and Vautheret, 2001). To date, several viruses (e.g., Potato virus X, PVX, Tobacco rattle virus, TRV, Tobacco mosaic virus, TMV and Tomato golden mosaic virus, TGMV) have been successfully used as vectors for VIGS in several dicotyledonous plants (Kumagai et al., 1995; Ruiz et al., 1998; Burton et al., 2000; Peele et al., 2001; Ratcliff et al., 2001; Liu, 2002; Hiriart et al., 2002) and one virus, Barley stripe mosaic virus (BSMV), in a monocotyledonous plant (barley) (Holzberg et al., 2002).
VIGS occurs in plants when there is sequence similarity between the virus sequence and a plant gene sequence, either native or transgenic (Lindbo et al., 1993; Kumagai et al., 1995). It has been indicated that the mechanism involved is post-transcriptional and targets RNA molecules in a sequence-specific manner (Smith et al., 1994; Goodwin et al., 1996; Guo and Garcia, 1997). Observations that viruses can both cause and be the targets of gene silencing have suggested that the mechanism is associated with anti-viral plant defense mechanisms (Pruss et al., 1997). Gene silencing can be activated in virally infected plants when part of a gene or its RNA is perceived as part of a virus genome or transcript. This can be achieved by including a portion or all of a plant gene sequence in a viral transcript.
While the foregoing studies have furthered the ability to use VIGS for analysis of plant gene function, the effectiveness of the technique has still been limited. The efficiency in delivery of VIGS vectors to plants of a number of different species in particular has been troublesome. In order to allow the analysis of large numbers of genes, efficient methods for the high-throughput analysis of plant gene function are needed. Such techniques would allow use of high-throughput VIGS for genome-wide identification of gene function where not previously possible.